Multiplex assay for the detection of citrus pathogens

ABSTRACT

The present invention provides methods and compositions for detecting multiple citrus pathogens using a multiplex branched DNA signal amplification reaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit to U.S. provisional applicationNo. 61/673,090, filed Jul. 18, 2012, which application is hereinincorporated by reference for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under Grant Nos.58-5302-1-119 and 58-5302-1-226, awarded by the U.S. Department ofAgriculture. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Citrus is susceptible to numerous disease caused by plant pathogens.There is a need for efficient and sensitive methods of detectingpathogens.

The method of the present invention provides a method for the detectionof nine citrus pathogens in a single sample using a multiplex branchedsignal amplification reaction. The present invention thus provides anaccurate, efficient, and quick method of detecting multiple citruspathogens that is also suitable for high throughput screenings.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods and kits for detecting up to ninecitrus pathogens where the pathogens are Citrus tristeza virus (CTV)universal, CTV genotype T30 and CTV genotype VT; Citrus psorosis virus(CPsV); Citrus tatter leaf virus (CTLV); Citrus leaf blotch virus(CLBV); Citrus exocortis viroid (CEVd); Hop stunt viroid (HSVd); andCitrus leprosis virus (CiLV). In some embodiments, the methods and kitsadditionally comprise components for detecting a housekeeping citrusgene, NADH dehydrogenase gene (Nad5) as an internal control.

In some aspects, the invention provides the following illustrativeembodiments:

Embodiment 1

A method for detecting the presence of at least one citrus pathogenselected from Citrus tristeza virus (CTV) universal, CTV genotype T30,CTV genotype VT, Citrus psorosis virus, Citrus tatter leaf virus, Citrusleaf blotch virus, Citrus exocortis viroid, Hop stunt viroid, or Citrusleprosis virus in a plant sample, the method comprising:

extracting RNA from said sample;performing a multiplex branched DNA signal amplification reaction;wherein the reaction comprises at least one capture extender probe andat least one label extender probe that targets the pathogen, wherein theat least one capture extender probe and the at least one label extenderprobe each comprises at least 8, 9, or 10 contiguous nucleotide of aprobe sequence shown in Table 1; anddetecting the presence or absence of a signal above background, whereinthe presence of the signal is indicative of the presence of thepathogen.

Embodiment 2

The method of embodiment 1, wherein the reaction comprises multiplecapture extender probes and multiple label extender probes that targetthe pathogen, wherein each of the multiple capture extender probes andmultiple label extender probes that target the pathogen comprises atleast 8, 9, or 10 contiguous nucleotides of a probe sequence as shown inTable 1.

Embodiment 3

The method of embodiment 1 or 2, wherein each of the probes that targetthe pathogen comprises a probe sequence set forth in Table 1.

Embodiment 4

The method of embodiment 1, further comprising detecting the presence orabsence of a second pathogen selected from Citrus tristeza virus (CTV)universal, CTV genotype T30, CTV genotype VT, Citrus psorosis virus,Citrus tatter leaf virus, Citrus leaf blotch virus, Citrus exocortisviroid, Hop stunt viroid, or Citrus leprosis virus in the plant sample,wherein the reaction comprises at least one capture extender probe andat least one label extender probe that target the second pathogen,wherein the capture extender probe and the label extender probe thattarget the second pathogen each comprise at least 8, 9, or 10 contiguousnucleotides of a probe sequence as shown in Table 1.

Embodiment 5

The method of embodiment 4, wherein the reaction comprises multiplecapture extender probes and multiple label extender probes that targetthe second pathogen, wherein each of the multiple capture extenderprobes and multiple label extender probes that target the secondpathogen comprise at least 8, 9, or 10 contiguous nucleotides of a probesequence as shown in Table 1.

Embodiment 6

The method of embodiment 4 or 5, wherein each of the probes that targetthe second pathogen comprises a probe sequence as shown in Table 1.

Embodiment 7

The method of embodiment 4, further comprising detecting the presence orabsence of a third, fourth, fifth, sixth, seventh, or eighth pathogenselected from Citrus tristeza virus (CTV) universal, CTV genotype T30,CTV genotype VT, Citrus psorosis virus, Citrus tatter leaf virus, Citrusleaf blotch virus, Citrus exocortis viroid, Hop stunt viroid, or Citrusleprosis virus in the plant sample, wherein the reaction comprises atleast one capture extender probe and at least one label extender probethat target the third, fourth, fifth, sixth, seventh, or eighthpathogen, wherein the capture extender probe and the label extenderprobe each comprise at least 8, 9, or 10 contiguous nucleotides of aprobe sequence as shown in Table 1.

Embodiment 8

The method of embodiment 7, wherein the reaction comprises multiplecapture extender probes and multiple label extender probes that targetthe third, fourth, fifth, sixth, seventh, or eighth pathogen, whereineach of the multiple probes comprises at least 8, 9, or contiguousnucleotides of a probe sequence as shown in Table 1.

Embodiment 9

The method of embodiment 7 or 8, wherein each of the probes that targetthe third, fourth, fifth, sixth, seventh, or eighth pathogen comprises aprobe sequence as shown in Table 1.

Embodiment 10

The method of embodiment 1, comprising detecting the presence or absenceof the nine pathogens Citrus tristeza virus (CTV) universal, CTVgenotype T30, CTV genotype VT, Citrus psorosis virus, Citrus tatter leafvirus, Citrus leaf blotch virus, Citrus exocortis viroid, Hop stuntviroid, and Citrus leprosis virus in the plant sample, wherein for eachof the nine pathogens, the reaction comprises a capture extender probeand a label extender probe, wherein each capture extender probe and eachlabel extender probe comprises at least 8, 9, or 10 contiguousnucleotides of a probe sequence as shown in Table 1.

Embodiment 11

The method of embodiment 10, wherein the reaction comprises multiplecapture extender probes and multiple label extender probes that targeteach of the nine pathogens, wherein each of the multiple captureextender probes and each of the multiple label extender probes comprisesat least 8, 9, or 10 contiguous nucleotides of a probe sequence shown inTable 1.

Embodiment 12

The method of embodiment 10 or 11, wherein each of the probes thattarget the nine pathogens comprises a probe sequence shown in Table 1.

Embodiment 13

The method of any one of embodiments 1 to 12, wherein the reactioncomprises at least one blocking probe listed in Table 1 for thecorresponding pathogen.

Embodiment 14

The method of any one of embodiment 1 to 12, wherein the reactioncomprises all of the blocking probes listed in Table 1.

Embodiment 15

The method of any one of embodiments 1 to 14, wherein the reactionfurther comprises using one of the capture extender probes and one ofthe label extender probes for Nad5 listed in Table 1.

Embodiment 16

The method of embodiment 15, wherein the reaction comprises using all ofthe label extender probes and all of the capture extender probes forNad5 listed in Table 1.

Embodiment 17

The method of any one of embodiments 1 to 16, wherein the plant sampleis from seed, foliage, limbs, trunk, bark, rootstock, fruit, germplasm,propagule, cuttings, or budwood.

Embodiment 18

A reaction mixture for detecting the presence of at least one citruspathogen selected from Citrus tristeza virus (CTV) universal, CTVgenotype T30, CTV genotype VT, Citrus psorosis virus, Citrus tatter leafvirus, Citrus leaf blotch virus, Citrus exocortis viroid, Hop stuntviroid, or Citrus leprosis virus in a plant sample, wherein the reactionmixture comprises at least one capture extender probe and at least onelabel extender probe that target the pathogen, where the at least onecapture extender probe and at least one label extender probe comprises8, 9, or 10 contiguous nucleotide of a probe sequence as shown in Table1.

Embodiment 19

The reaction mixture of embodiment 18, wherein the reaction comprisesmultiple capture extender probes and multiple label extender probes thattarget the pathogen, wherein each of the multiple capture extenderprobes and multiple label extender probes comprises at least 8, 9, or 10contiguous nucleotides of a probe sequence as shown in Table 1.

Embodiment 20

The reaction mixture of embodiment 18 or 19, wherein each of the probesthat target the pathogen comprises a probe sequence set forth in Table1.

Embodiment 21

The reaction mixture of embodiment 18, further comprising at least onecapture extender probe and at least one label extender probe that targeta second pathogen selected from Citrus tristeza virus (CTV) universal,CTV genotype T30, CTV genotype VT, Citrus psorosis virus, Citrus tatterleaf virus, Citrus leaf blotch virus, Citrus exocortis viroid, Hop stuntviroid, or Citrus leprosis virus, wherein the capture extender probe andthe label extender probe that target the second pathogen each compriseat least 8, 9, or 10 contiguous nucleotides of a probe sequence as shownin Table 1.

Embodiment 22

The reaction mixture of embodiment 21, wherein the reaction comprisesmultiple capture extender probes and multiple label extender probes thattarget the second pathogen, wherein each of the multiple captureextender probes and multiple label extender probes comprises at least 8,9, or 10 contiguous nucleotides of a probe sequence as shown in Table 1.

Embodiment 23

The reaction mixture of embodiment 21 or 22, wherein each of the probesthat target the second pathogen comprises a probe sequence as shown inTable 1.

Embodiment 24

The reaction mixture of embodiment 21, further comprising at least onecapture extender probe and at least one label extender probe that targeta third, fourth, fifth, sixth, seventh, or eighth pathogen selected fromCitrus tristeza virus (CTV) universal, CTV genotype T30, CTV genotypeVT, Citrus psorosis virus, Citrus tatter leaf virus, Citrus leaf blotchvirus, Citrus exocortis viroid, Hop stunt viroid, or Citrus leprosisvirus, wherein the capture extender probe and the label extender probeeach comprise at least 8, 9, or 10 contiguous nucleotides of a probesequence as shown in Table 1.

Embodiment 25

The reaction mixture of embodiment 24, wherein the reaction comprisesmultiple capture extender probes and multiple label extender probes thattarget the third, fourth, fifth, sixth, seventh, or eighth pathogen,wherein each of the multiple probes comprises at least 8, 9, or 10contiguous nucleotides of a probe sequence as shown in Table 1.

Embodiment 26

The reaction mixture of embodiment 24 or 25, wherein each of the probesthat target the third, fourth, fifth, sixth, seventh, or eighth pathogencomprises a probe sequence as shown in Table 1.

Embodiment 27

The reaction mixture of embodiment 18, comprising a capture extenderprobe and a label extender probe for each of the nine pathogens Citrustristeza virus (CTV) universal, CTV genotype T30, CTV genotype VT,Citrus psorosis virus, Citrus tatter leaf virus, Citrus leaf blotchvirus, Citrus exocortis viroid, Hop stunt viroid, and Citrus leprosisvirus, wherein each capture extender probe and each label extender probecomprises at least 8, 9, or 10 contiguous nucleotides of a probesequence as shown in Table 1.

Embodiment 28

The reaction mixture of embodiment 27, comprising multiple captureextender probes and multiple label extender probes that target each ofthe nine pathogens, wherein each of the multiple capture extender probesand each of the multiple label extender probes comprises at least 8, 9,or 10 contiguous nucleotides of a probe sequence shown in Table 1.

Embodiment 29

The reaction mixture of embodiment 27 or 28, wherein each of the probesthat target the nine pathogens comprises a probe sequence shown in Table1.

Embodiment 30

The reaction mixture of any one of embodiments 18 to 29, wherein thereaction comprises at least one blocking probe listed in Table 1 for thecorresponding pathogen.

Embodiment 31

The reaction mixture of any one of embodiments 18 to 29, wherein thereaction mixture comprises all of the blocking probes listed in Table 1.

Embodiment 32

The reaction mixture of any one of embodiments 18 to 31, wherein thereaction mixture further comprises one of the capture extender probesand one of the label extender probes for Nad5 listed in Table 1, or allof the capture extender probes and all of the label extender probes forNad5 listed in Table 1.

Embodiment 33

A kit mixture for detecting the presence of at least one citrus pathogenselected from Citrus tristeza virus (CTV) universal, CTV genotype T30,CTV genotype VT, Citrus psorosis virus, Citrus tatter leaf virus, Citrusleaf blotch virus, Citrus exocortis viroid, Hop stunt viroid, or Citrusleprosis virus in a plant sample, wherein the kit comprises at least onecapture extender probe and at least one label extender probe that targetthe pathogen, where the at least one capture extender probe and at leastone label extender probe comprises 8, 9, or 10 contiguous nucleotide ofa probe sequence as shown in Table 1.

Embodiment 34

The kit of embodiment 33, wherein the kit comprises multiple captureextender probes and multiple label extender probes that target thepathogen, wherein each of the multiple capture extender probes andmultiple label extender probes comprises at least 8, 9, or 10 contiguousnucleotides of a probe sequence as shown in Table 1.

Embodiment 35

The kit of embodiment 33 or 34, wherein each of the probes that targetthe pathogen comprises a probe sequence set forth in Table 1.

Embodiment 36

The kit of embodiment 33, further comprising at least one captureextender probe and at least one label extender probe that target asecond pathogen selected from Citrus tristeza virus (CTV) universal, CTVgenotype T30, CTV genotype VT, Citrus psorosis virus, Citrus tatter leafvirus, Citrus leaf blotch virus, Citrus exocortis viroid, Hop stuntviroid, or Citrus leprosis virus, wherein the capture extender probe andthe label extender probe that target the second pathogen each compriseat least 8, 9, or 10 contiguous nucleotides of a probe sequence as shownin Table 1.

Embodiment 37

The kit of embodiment 36, wherein the reaction comprises multiplecapture extender probes and multiple label extender probes that targetthe second pathogen, wherein each of the multiple capture extenderprobes and multiple label extender probes comprises at least 8, 9, or 10contiguous nucleotides of a probe sequence as shown in Table 1.

Embodiment 38

The kit of embodiment 36 or 37, wherein each of the probes that targetthe second pathogen comprises a probe sequence as shown in Table 1.

Embodiment 39

The kit of embodiment 36, further comprising at least one captureextender probe and at least one label extender probe that target athird, fourth, fifth, sixth, seventh, or eighth pathogen selected fromCitrus tristeza virus (CTV) universal, CTV genotype T30, CTV genotypeVT, Citrus psorosis virus, Citrus tatter leaf virus, Citrus leaf blotchvirus, Citrus exocortis viroid, Hop stunt viroid, or Citrus leprosisvirus, wherein the capture extender probe and the label extender probeeach comprise at least 8, 9, or 10 contiguous nucleotides of a probesequence as shown in Table 1.

Embodiment 40

The kit of embodiment 39, wherein the kit comprises multiple captureextender probes and multiple label extender probes that target thethird, fourth, fifth, sixth, seventh, or eighth pathogen, wherein eachof the multiple probes comprises at least 8, 9, or 10 contiguousnucleotides of a probe sequence as shown in Table 1.

Embodiment 41

The kit of embodiment 39 or 40, wherein each of the probes that targetthe third, fourth, fifth, sixth, seventh, or eighth pathogen comprises aprobe sequence as shown in Table 1.

Embodiment 42

The kit of embodiment 33, comprising a capture extender probe and alabel extender probe for each of the nine pathogens Citrus tristezavirus (CTV) universal, CTV genotype T30, CTV genotype VT, Citruspsorosis virus, Citrus tatter leaf virus, Citrus leaf blotch virus,Citrus exocortis viroid, Hop stunt viroid, and Citrus leprosis virus,wherein each capture extender probe and each label extender probecomprises at least 8, 9, or 10 contiguous nucleotides of a probesequence as shown in Table 1.

Embodiment 43

The kit of embodiment 42, comprising multiple capture extender probesand multiple label extender probes that target each of the ninepathogens, wherein each of the multiple capture extender probes and eachof the multiple label extender probes comprises at least 8, 9, or 10contiguous nucleotides of a probe sequence shown in Table 1.

Embodiment 44

The kit of embodiment 42 or 43, wherein each of the probes that targetthe nine pathogens comprises a probe sequence shown in Table 1.

Embodiment 45

The kit of any one of embodiments 33 to 44, wherein the kit comprises atleast one blocking probe listed in Table 1 for the correspondingpathogen.

Embodiment 46

The kit of any one of embodiments 33 to 44, wherein the kit comprisesall of the blocking probes listed in Table 1.

Embodiment 47

The kit of any one of embodiments 33 to 46, wherein the kit furthercomprises one of the capture extender probes and one of the labelextender probes for Nad5 listed in Table 1, or all of the captureextender probes and all of the label extender probes for Nad5 listed inTable 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides illustrative data showing a 10-Plex reaction detectingcitrus pathogens.

FIG. 2 provides illustrative data showing universal andgenotype-specific detection of citrus pathogen CTV.

FIG. 3 provides illustrative data showing specific detection of citrusviroids CEVd and HSVd.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, a “probe that targets a pathogen” refers to a nucleotidesequence that hybridizes to a desired region of a target nucleic acid inthe pathogen.

The term “hybridization” refers to the formation of a duplex structureby two single stranded nucleic acids due to complementary base pairing.Hybridization can occur between exactly complementary nucleic acidstrands or between nucleic acid strands that contain minor regions ofmismatch. As used herein, the term “substantially complementary” refersto sequences that are complementary except for minor regions ofmismatch. Typically, the total number of mismatched nucleotides over ahybridizing region is not more than 3 nucleotides for sequences about 15nucleotides in length.

The term “probe” refers to an oligonucleotide that selectivelyhybridizes to a target nucleic acid under suitable conditions. Ahybridization assay carried out using a probe under sufficientlystringent hybridization conditions enables the selective detection of aspecific target sequence comprising the region of interest of a pathogennucleic acid. The probe hybridizing region is preferably from about 10to about 35 nucleotides in length. In some embodiments, the probehybridizing region is 15 to about 35 nucleotides in length. The use ofmodified bases or base analogues which affect the hybridizationstability, which are well known in the art, may enable the use ofshorter or longer probes with comparable stability.

The term “target sequence” or “target region” refers to a region of anucleic acid in a pathogen of interest to which a probe to the pathogenbinds.

A “capture extender probe” or “CE probe” as used here is apolynucleotide that is capable of hybridizing to a nucleic acid ofinterest from a citrus pathogen and to a capture probe. The captureextender probe has a first polynucleotide sequence that is complementaryto a capture probe, and a second polynucleotide sequence, e.g., asequence as shown in Table 1, which is complementary to a citruspathogen nucleic acid as described herein. The capture probe istypically immobilized to a solid support, including but not limited to achip (e.g., an array), well, bead, or other solid support or matrix.

A “label extender probe” or “LE” as used here is a polynucleotide thatis capable of hybridizing to a nucleic acid of interest from a pathogenand to a label probe system. The label extender probe has a firstpolynucleotide sequence that is complementary to a polynucleotidesequence of the label probe system and a second polynucleotide sequence,e.g., a sequence as shown in Table 1, which is complementary to a citruspathogen as described herein. The signal-amplifying probe in the presentinvention typically comprises branched DNA, e.g., may include apre-Amplifier probe, an Amplifier probe, and a Label probe.

INTRODUCTION

The present invention provides methods to diagnose infection with citruspathogens. In some embodiments, the methods can be used inhigh-throughput screenings of thousands of plant samples in regulatoryand research programs. Branched DNA technology (bDNA) employs a sandwichnucleic acid hybridization assay for nucleic acid detection andquantification that amplifies the reporter signal rather than the targetsequence of interest that is to be detected. Thus, bDNA technologyamplifies signal directly from captured target RNA without purificationor reverse transcription. RNA quantitation is performed directly from atissue sample. By measuring the nucleic acid at the sample source, theassay avoids variations or errors inherent to extraction andamplification of target polynucleotides. The QuantiGene Plex technologycan be combined with multiplex bead based assay system such as theLuminex system described below to enable simultaneous detection ofmultiple pathogens of interest.

In brief, in an assay of the invention, a target nucleic acid to bedetected is released from cells and captured by a Capture Probe (CP) ona solid surface (e.g., a well of a microtiter plate) through syntheticoligonucleotide probes called Capture Extenders (CEs). Each captureextender has a first polynucleotide sequence that can hybridize to thetarget nucleic acid and a second polynucleotide sequence that canhybridize to the capture probe. Typically, two or more capture extendersare used. Probes of another type, called Label Extenders (LEs),hybridize to different sequences on the target nucleic acid and tosequences on an amplification multimer. Additionally, Blocking Probes(BPs), which hybridize to regions of the target nucleic acid notoccupied by CEs or LEs, are typically used to reduce non-specific targetprobe binding. A probe set for a given nucleic acid thus has CEs, LEs,and typically BPs for the target citrus pathogen. The CEs, LEs, and BPsare complementary to nonoverlapping sequences in the target nucleic acidfrom the citrus pathogen, and are typically, but not necessarily,contiguous.

Signal amplification begins with the binding of the LEs to the targetmRNA. An amplification multimer is then typically hybridized to the LEs.The amplification multimer has multiple copies of a sequence that iscomplementary to a label probe (it is worth noting that theamplification multimer is typically, but not necessarily, abranched-chain nucleic acid; for example, the amplification multimer canbe a branched, forked, or comb-like nucleic acid or a linear nucleicacid). A label, for example, alkaline phosphatase, is covalentlyattached to each label probe. (Alternatively, the label can benoncovalently bound to the label probes.) In the final step, labeledcomplexes are detected, e.g., by the alkaline phosphatase-mediateddegradation of a chemilumigenic substrate, e.g., dioxetane. Luminescenceis reported as relative light unit (RLUs) on a microplate reader. Theamount of chemiluminescence is proportional to the level of mRNAexpressed from the target gene.

The present invention provides a method and compositions for detectingthe presence or absence of at least one, at least two, at least three,at least four, at least five, at least six, at least seven, at leasteight, or all nine of the citrus pathogens described herein. Asexplained above, detection is performed using bDNA signal amplificationtechnology and capture extender probes and label extender probes thattarget the pathogen nucleic acid regions described herein. The generaldesign of branched amplification assays, e.g., suitable amplificationmultimers, considerations in designing capture probes that bind to thecapture extenders, etc.; configuration; and hybridization conditions forsuch reactions can be determined using methods known in the art (see,e.g., U.S. Patent Application Publication No. 20120003648 and thereferences cited therein).

Citrus Pathogen Probes

The nine pathogen targets and internal citrus gene were developed basedon specific genomic sequences and characteristics in the pathogens'genome. The probes (Table 1) included Capture Extenders (CE), LabelExtenders (LE), Blocking Probes (BL) as per manufacturer'srecommendations were designed and developed based on the conservedsequences in the genome of each pathogen. CTV-Pan (universal) probes wasdesigned based on the pathogen sequence alignment of CTV isolatesworldwide and included all major CTV genotypes. CTV-T30 (mild strain)probes were designed based on the sequence alignment of worldwide T30genotype isolates. CTV-VT probes were designed based on sequencealignment of worldwide CTV isolates having a VT genotype. Similarly,probes for CPV, CTLV, CLBV, CEVd, HSVd and CiLV were based on pathogensequence alignment from data deposited in GenBank. In the presentinvention, the CE and LE probe sequences shown in Table 1 are theregions of the CE and LE oligonucleotides that target the pathogen.

The present invention employs CE and LE probes that comprise sequencespresented in Table 1 or are variants of the sequences in Table 1 thatretain the ability to hybridize to the same target nucleic acid sequenceas the probes shown in Table 1 such that the presence of the pathogen ina plant sample can be detected. Such variant probe sequences typicallyhave no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide changes relativeto a probe sequence as shown in Table 1. In some embodiments, a variantprobe useful in the invention comprises at least 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20, or more, contiguous nucleotides of asequence shown in Table 1.

The methods and compositions for detecting one or more of the 9 citruspathogens as described herein may also include probe to detect a controlnucleic acid sequence. In some embodiments, the control is ahousekeeping gene that is common to citrus plants. In some embodiments,the housekeeping gene is NADH dehydrogenase gene. In some embodiments,the CE and LE probes comprises the NAD sequences shown in Table 1 or arevariants of the sequences that retain the ability to hybridize to thetarget region in the NAD sequence. In some embodiments, such a variantprobe comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, or more, contiguous nucleotides of a sequence shown in Table1; or has no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide changesrelative to the sequence shown in Table 1.

In some embodiments, one or more blocking probes may be employed. Table1 provides examples of sequences that may be employed in blocking probesuseful in the invention.

In some embodiments, multiple capture extender and/or label extenderprobes shown in Table 1, or variants thereof as described herein, areused in the invention. In some embodiments, each of the probes thattarget a pathogen is shown in Table 1. In some embodiments, all of theprobes, including any blocking probes, shown in Table 1, or variantsthereof as described herein, for a given pathogen are used in theinvention.

Plant Samples

The sample evaluated for the presence of citrus pathogens can be fromany plant material (e.g., seed, foliage, limbs, trunk, bark, rootstock,fruit, germplasm, propagule, cuttings, and budwood). RNA is extractedusing well known techniques. Such an RNA sample may also comprise DNA.Methods for extracting RNA from a plant samples are known to thoseskilled in the art and are described in Bilgin et al., Nature Protocols,4:333-340, (2009); Berendzen et al., Plant Methods, 1:4 (2005); ElspethMacRae, Methods in Molecular Biology, vol. 353: Protocols for NucleicAcid Analysis by Nonradioactive Probes, Second Edition, Humana Press,New Jersey, 15-24, (2007). Non-limiting examples of commerciallyavailable plant RNA extraction kits include include RNAeasy Plant MiniKit (Qiagen, Hilden, Germany), PrepEase Plant Spin Kit (Affymetrix,Santa Clara, Calif.), Agilent Plant RNA Isolation Mini Kit (AgilentTechnologies, Santa Clara, Calif.), Plant RNA Isolation Aid (Ambion,Austin, Tex.), and Spectrum Plant total RNA kit (Sigma-Aldrich, St.Louis, Mo.).

The following examples are offered to illustrate, but not to limit, theclaimed invention.

EXAMPLES Example 1 High Throughput Assays for Rapid and Accurate 10-PlexDetection of Citrus Pathogens

The assay was developed using a QuantiGene Plex and Luminex based assayprocedure. The probes used for detecting the pathogens are shown inTable 1.

TABLE 1Sequence of specific probes including Capture Extenders (CE), Label Extenders (LE), Blocking Probes (BL) designed and developed for thespecific detection of nine citrus pathogen targets and a housekeeping citrusgene as an internal control with QuantiGene Plex and Luminex based assay.Targets^(a) Probes Probe sequence (5′→3′) CTV-Pan CTV001 CEctccgcctgaaacactcagactc (all major CTV CTV002 CE atgaagtggtgttcacggagaagenotypes) CTV003 CE attttaaagactttacccatccggt CTV004 CEggttcacgcatacgttaagcc CTV005 CE aacacacactctaaggagaacttcttt CTV006 CEcccccatagggacagtgtgttgg CTV007 LE gaacttattccgtccacttcaatcag CTV008 LEaagggtttttaccaacccgacata CTV009 LE tattgtctagtgatacatcaccatcat CTV010 LEatatggttaattttcccctcgatc CTV011 LE gggagcttagaccaacgagagg CTV012 LEtcacttgagaccactaccactctgt CTV-T30 (CTV- T30001 CE cgggtgaatttgaatcgaaattT30 genotype) T30002 CE ggatcgagctccggagata T30003 CE ccaagtcccgcagggtccT30004 CE aaccgtctggttgggatttaca T30005 CE tgtattgatattatgggcgtagaacT30006 CE aagggacgatcggcccagcagcc T30007 CE catacctccaagcgcccgcaaT30008 CE tggggactttcacgcacagt T30009 LE agcgaaagtcgaggacttgaa T30010 LEcgaaattatgtaatcgctgcgtac T30011 LE aggcgcgccagatgcg T30012 LEtgcaggactccaacggtattaa T30013 LE ggttgtatcagtgccgaagaag T30014 LEgaaaactccttaaccaccgtagt T30015 LE ttaatcgcgcgaacagca T30016 LEaataggacgtccggcagct T30017 LE cggagcgcggagcgtc T30018 LEggaggccacagaggcatc T30019 LE acaccagatgtgtcgaaaacag T30020 LEcagagcggggacgcacg T30023 LE tcttcgccttgcgaatgga T30024 LEggctgagaaagaatgcagaatctt T30025 LE cgagagaagagagaagaagccc T30026 LEgtgccgcaagggacttcc T30027 LE gcctgcgaagtctgtgacgc T30028 LEagggtcaactagtttcgcaacac T30029 BL gctagctccgagtttcgacatat T30031 BLgcgcgaactgagaacgga CTV-VT (CTV- VT002 CE ggacgtgatttccggagggVT genotype) VT003 CE accgattcccgcagcgt VT004 CE ggcaatttgccgggatttacVT005 CE atttgtttgtatgggcgtagtg VT007 CE agaatacctccaaatgcccg VT008 CEggcgtcccttaagtttgatct VT009 LE gaaagtcaaggacttgaagcg VT010 LEtgatgtaatcgctgcgtacagc VT011 LE gcgccagatgcgcgaga VT012 LEgactccaacggtgttaaaggc VT013 LE ggttgtttcagtaccgaagaagt VT014 LEaaaattccttaaccaccttggt VT015 LE ttaatcgcgcgaacagca VT016 LEtgggacgtccggcagct VT017 LE agagcgcggagcgtcaa VT018 LEggaggccacagaggcatcc VT019 LE cgacaccagatgtgtcgataacag VT020 LEcgacagagcggggacgta VT021 LE gcagtaaggggaggtttacacag VT022 LEtggacttcttggcggcg VT023 LE tctttcttcgccttgcgaa VT024 LEccggctaagaaagaaagcagaa VT025 LE cgtgccgcaggggactt VT026 LEgcgcctacgaagtctatgacg VT027 LE atggtagggtctactcgtttcataac VT028 LEcgtcttggggactctcgtgc VT029 BL agctccgagtttcgacatgttat VT030 BLaaacaggatttccgtagaggg VT031 BL gcgcgaacagaaaacgga VT032 BLcccgtgagaagagtgaagaagc Psorosis (CPsV) psoro001 CE tggcgatggtgaagggccpsoro002 CE aagaacaaggggtttcagaatgatag psoro003 CE agcctcactccagatggcagapsoro004 CE tcaattgcaataagagattttctgaa psoro005 CEctcctgaatccctgatgccatt psoro006 CE atctgtgagatatgctgggtttgc psoro007 CEaacaaagaaattccctgcaaggg psoro008 CE gtgaggaattgagccatgctcc psoro009 LEcatggagtgtgttgacaaaacca psoro010 LE attgacatggccgagaggataataapsoro011 LE aaaaggcttcatcctttatctgatga psoro012 LEtggagggacaatggaagaatcag psoro013 LE gctggaaaccaatcaaaagattgaaaaapsoro014 LE gtcccctgctgttggtgcaa psoro015 LEtgtttctcaagattgatatagacaactt psoro016 LE gaagctgtatgatggtgatgtaagtttCTLV CTLV001 CE tgctgagagggacctaaatcctct CTLV002 CEgggaggaaccgtcagaagttcc CTLV003 CE gtgattgcagagaagaaggtaaagctc CTLV004 CEaagaaattcacgagccaaatcagc CTLV005 CE caaaagctttgggccatttctt CTLV006 CEcctgcctcgaaaaccccttt CTLV007 LE tgttgaagcacgtcttccaaactcat CTLV008 LEgaaactgggtcttatcagatgaccc CTLV009 LE agaagtagcagcaaaggttttcaattcCTLV010 LE ttgtccttcagtacgaaaaagcct CTLV011 LE cgactcctaaccctccagttccaCTLV012 LE cctgcaagaccgcgaccaa CTLV013 LE ttaagtataaaggcaggcatgtcaa CLBVCLBV001 CE cagctctgaattttcgaatgatgtca CLBV002 CEttgagtgactcattcaattcttcaa CLBV003 CE cccagccaaaattgcagct CLBV004 CEatctttgggaaatgtctttcaact CLBV005 CE gctcatgccctttttttttcaaatt CLBV006 LEtgaggaatggttcaactatggct CLBV007 LE tcagaatttctgtcctcatcagatg CLBV008 LEtctccatgctcggccacta CLBV009 LE tcagggtccacctcctctgtg CLBV010 LEtgtgatctcaagctgtgatgcat CLBV011 LE ttcaagctgctgctctctatctgc CLBV012 LEccactgccggtcagtggtt CLBV013 LE cagcatgtaccggttcagaagat CLBV014 LEactgtggagcgtgtgctgatt CLBV015 LE gcagatcattcaccacatgca CLBV016 BLggaaaaaatggcgaagaagacc CLBV017 BL ctttttttgaataaactctgccgtac CLBV018 BLtgtttctcagatcttctgcttctgc CEVd CEVd001 CE tcttttttcttttcctgcctgcCEVd002 CE ggatccctgaaggacttctt CEVd003 CE tcctccaggtttccccgg CEVd004 CEttctccgctggacgccag CEVd005 CE cctcgcccggagagcagt CEVd006 CEtagggttccgagggctttcac CEVd007 LE ggaacctcaagaaagatcccg CEVd008 LEagggtcaggtgagcaccaca CEVd009 LE cccccccgacctcgact CEVd010 LEtgatccgcggcgaccg CEVd011 LE aaaggaaggagacgagctcctgt CEVd012 LEggatgtggagccagcagcg CEVd013 LE tcagttgtttccaccgggtagt CEVd014 LEgcggtttggggttgaagct HSVd CVdII001 CE ttttctttgcttgcccatgc CVdII002 CEggattctgagaagagttgcccc CVdII003 CE agctagaagcctctactccagagc CVdII004 CEggacgatcgatggtgtttcga CVdII005 CE agccaggagaaggtaaaagaagaag CVdII006 CEcgaaccgagaggtgatgcca CVdII007 LE ggcaactcgagaattccccag CVdII008 LEggggctcctttctcaggtaagt CVdII009 LE accgcggccctctctcc CVdII010 LEccggtcgcgtctcatcgga CVdII011 LE ggcagaggctcagatagacaaaaa CVdII012 LEgggctcaagagaggatccgc Leprosis (CiLV) leprosis3|CEtgctaatatcacgcagaccttca leprosis9|CE ggccttctgcttagcaggttt leprosis11|CEtgggtggagcaagctgctt leprosis14|CE cggcatattttgggcagtg leprosis18|CEgcttccattaccttaaaatcaggta leprosis19|CE gacggcaactaggtcctcagaaleprosis1|LE ctcaatggcctgcataatctca leprosis2|LE ggaacagacacgttgtgccgleprosis4|LE actgctgcttcttcttagtaggct leprosis5|LE gtgacagttgttgaggttgcgleprosis7|LE ccgggttgcagttgctgag leprosis8|LE cttggcctgataaccactaggaleprosis16|LE ttataatatgtcatccctatctgcttc leprosis17|LEacgcatagggctcggatatc leprosis21|LE atactatataagcgcttctcaaagctleprosis22|LE gtcgcttcgggaagccc leprosis23|LE ccgggacaacgttctttatggleprosis24|LE caatgtagtgatcactgaactcgaata Nad5 Nad54|CEggtcattatagcggttccttctga Nad55|CE gaagagaatgaaacgcacgtagt Nad59|CEcaaacatttccgatgagatcca Nad515|CE aataacacataaatcgagggctatg Nad519|CEaaatatgaagcaagacctactccct Nad522|CE ctcgattgacaggcatagcttt Nad51|LEgggcaaaaatacgataagtagataca Nad52|LE ctgctacggaactaccgagaag Nad56|LEtcataaaaagcaatcagagataagatc Nad57|LE actagctcccggtgcgact Nad510|LEcaagaagccccaagaagcat Nad511|LE actacggtcgggctatcgaa Nad512|LEcttatggatgtaaccacaattaacatc Nad513|LE tggaataaagatggaccaagcta Nad517|LEgagagttatctccagtcaccaacat Nad518|LE cccatcccaggaataattgaa Nad520|LEgtcgtgtaaaccagaaatgaattaac Nad521|LE tgtagctgctttatctgcctgaa^(a)CTV-Pan: Citrus tristeza virus major genotypes (universal), CTV-T30:CTV genotype T30, CTV-VT: CTV genotype VT, Psorosis: Citrus psorosisvirus (CPsV), CTLV: Citrus tatter leaf virus, CLBV: Citrus leaf blotchvirus, CEVd: Citrus exocortis viroid, HSVd: Hop stunt viroid (syn.citrus viroid IIa, IIb, and IIc), Leprosis: Citrus leprosis virus(CiLV), Nad5: NADH dehydrogenase gene, a housekeeping citrus gene, usedas an internal control.

The procedure from the QuantiGene Plex 2.0 Assay User Manual fromAffymetrix/Panomics Inc is shown below:

Capturing Target RNA from Total, Purified, In Vitro Transcribed RNA orTotal Nucleic Acid1. Sample and reagent preparation: thaw probe set, blocking reagent andtotal nucleic acid samples (both RNA and DNA or RNA only), and placethem on ice.2. Pre-warm lysis mixture at 37° C. for 30 minutes.3. Prepare a working bead mix including nuclease-free water, lysismixture, blocking reagent, capture beads, probe set, according toreaction composition (Table 2).

TABLE 2 Working Bead Mix Set Up Order of addition Reagent Per well (μl)1 Nuclease-free water 38.7 2 Lysis mixture 33.3 3 Blocking reagent 2 4Capture beads 1 5 Probe set 5 Total 804. Vortex working bead mix for 30 sec, transfer 80 μl to each well ofthe hybridization plate.5. Add 20 μl nucleic acid samples (or RNA sample) to each well of theabove plate.6. Seal the hybridization plate with a pressure seal and mount the plateinto the shaking incubator.7. Incubate for 18-22 hours at 54° C. at 600 rpm.

Signal Amplification and Detection of RNA Targets

1. Place label probe diluent and SAPE diluent to room temperature.Incubate amplifier diluent at 37° C. for 20 minutes.2. Prepare 200 ml wash buffer including 0.6 ml wash buffer Component 1,10 ml wash buffer Component 2 and 189.4 ml nuclease-free water.3. Add 36 μl pre-amplifier to 12 ml amplifier diluent.4. Take the hybridization plate out of the shaking incubator, and spinat 240 g for 60 seconds.5. Open the pressure seal, mix with pipette, then transfer thehybridization mixture to the magnetic separation plate.6. Put the magnetic separation plate on the plate holder of the platewasher for 60 seconds, then empty the magnetic separation and wash threetimes with 100 μl wash buffer.7. Add 100 μl pre-amplifier solution to each well.8. Seal the magnetic separation plate with a foil plate seal and shakefor 60 minutes at 50° C. with 600 rpm.9. Add 36 μl amplifier to 12 ml amplifier diluent.10. Take the magnetic separation plate out of the shaking incubator.11. Open the foil plate seal.12. Put the magnetic separation plate on the plate holder of the platewasher for 60 seconds, then empty the magnetic separation plate and washthree times with 100 μl wash buffer.13. Add 100 μl amplifier solution to each well.14. Seal the magnetic separation plate with a foil plate seal and shakefor 60 minutes at 50° C. with 600 rpm.15. Add 36 μl label probe to 12 ml label probe diluent.16. Take the magnetic separation plate out of the shaking incubator andopen the foil plate seal.17. Put the magnetic separation plate on the plate holder of the platewasher for 60 seconds, then empty the magnetic separation plate and washthree times with 100 μl wash buffer.18. Add 100 μl label probe solution to each well.19. Seal the magnetic separation plate with a foil plate seal and shakefor 60 minutes at 50° C. with 600 rpm.20. Add 36 μl SAPE to 12 ml SAPE diluent.21. Take the magnetic separation plate out of the shaking incubator andopen the foil plate seal.22. Put the magnetic separation plate on the plate holder of the platewasher for 60 seconds, then empty the magnetic separation plate and washthree times with 100 μl wash buffer.23. Add 100 μl SAPE solution to each well.24. Seal the magnetic separation plate with a foil plate seal and shakefor 30 minutes at 50° C. with 600 rpm.25. Take the magnetic separation plate out of the shaking incubator,open the foil plate seal.26. Put the magnetic separation plate on the plate holder of the platewasher for 60 seconds, then empty the magnetic separation plate and washthree times with 100 μl SAPE wash buffer.27. Add 130 μl SAPE wash buffer to each well.28. Seal the magnetic separation plate with a foil plate seal and coverthe magnetic separation plate with foil and shake for 2-3 minutes atroom temperature with 600 rpm, then use Luminex instrument to read.

Initially, the assay was performed using nine samples from healthy andinfected citrus plants with CTV genotype T30, CTV genotype VT, CPsV(Psorosis), CTLV, CLBV, CEVd, HSVd, CiLV (Leprosis), respectively (FIG.1). A procedure for high throughput robotic extraction and purificationof nucleic acid targets, optimized for citrus tissues, was developed andused with the Luminex-based QuantiGene Plex system to increaseuniformity and cost effectiveness of the test. Sample CTV-T30 wasdetected to show positive reactions with both CTV-Pan and CTV-T30, butnot CTV-VT and other pathogen targets. In addition, sample CTV-T30 wasdetected to show positive reactions with Nad5, the positive internalcontrol for citrus plants, which could be used to access the RNAextraction quality and to normalize data for accurate quantification ofthe pathogen populations among samples. Sample CTV-VT was confirmed tohave CTV genotype VT and HSVd from other studies. In contrast to sampleCTV-T30, CTV-VT was detected to show positive reactions with Nad5,CTV-Pan, CTV-VT, and HSVd, but not CTV-T30 and the other RNA pathogentargets. Sample HSVd showed positive reactions with Nad5 and HSVd, butnot other pathogen targets. Similarly, samples CEVd, CPsV and CTLVshowed positive reactions with Nad5 and their pathogen targets,respectively, but not other pathogen targets. Sample CLBV was confirmedto have HSVd and CLBV from other studies. In the assay, sample CLBVshowed positive reactions with Nad5, CLBV and HSVd, but not otherpathogen targets. Sample Leprosis was confirmed to have CTV, HSVd andPsorosis from other studies. In the assay, sample Leprosis showedpositive reactions with Leprosis, Nad5, CTV-Pan, CTV-T30, CTV-VT, andHSVd. Finally, the healthy Navel sweet orange control sample showedpositive reaction with Nad5 but not to any pathogen targets. These datashowed that the assay is capable of specific detection of each targetincluding Nad5, CTV-Pan, CTV genotype T30, CTV genotype VT, Psorosis,CTLV, CLBV, CEVd, HSVd, and Leprosis.

Developmental assays were performed using more CTV samples from healthyand infected citrus plants (FIG. 2). Every sample tested had a positivereaction with Nad5 indicating that nucleic acid extracted per plant waseffective. In addition, sample CTV-1 containing CTV genotype T30 showedpositive reactions with both CTV-Pan and CTV-T30, but not CTV-VT. SampleCTV-2 containing CTV genotype VT showed positive reactions with bothCTV-Pan and CTV-VT, but not CTV-T30. Sample CTV-3 containing CTVgenotype T36 showed positive reaction with CTV-Pan only, but not toCTV-T30 or CTV-VT. Lastly, samples 4 and 5 containing both T30 and VTgenotypes showed positive reactions with CTV-Pan, CTV-T30 and CTV-VT.These data further validate the 10-plex detection system could detectbroad-spectrum CTV strains but also the major genotypes T30 and VT.

The assay was also performed using healthy citrus and citrus infectedwith citrus viroids (FIG. 3). There are seven known distinct viroidspecies representing four genera in the Pospiviroidae family. They are:Citrus exocortis viroid (CEVd, genus Pospiviroid), Hop stunt viroid(HSVd, genus Hostuviroid), Citrus bark cracking viroid (CBCVd, genusCocadviroid) and Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid(CDVd), Citrus viroid V (CVd-V) and CVd-VI of the genus Apscaviroid.Collectively, these various citrus viroids cause abnormal growth. Allsamples tested were positive for Nad5 and demonstrated good qualitynucleic acid extraction and purification. Viroid-3, -5 and -7 containedHSVd and showed positive reaction with HSVd but not CEVd. Viroid-6contained CEVd and showed a positive reaction with CEVd but not HSVd.Samples infected with known multiple viroid species were also tested inthe assay. Sample viroid-2 contained CEVd and CBCVd and showed apositive reaction with CEVd, but not HSVd. Sample viroid-4 containingCBLVd, HSVd, CDVd, CBCVd, CVd-V and CEVd showed positive reactions withboth CEVd and HSVd. In short, it was further validated that the 10-plexdetection system could detect specifically different citrus viroidspecies CEVd and HSVd, respectively.

The high throughput robotic extraction and purification of nucleic acidtargets, optimized for citrus tissues, it showed that samples obtainedfrom fresh or frozen tissue crude extraction using QuantiGene sampleprocessing kit were consistently detected. Finally, sensitivity studiesusing serial dilutions of CTV and HSVd samples, respectively, suggestedthat those samples, obtained by the high throughput robotic extractionand purification of nucleic acid targets, were consistently detectedafter dilution of up to 1000 times.

All publications, patents, accession numbers, and patent applicationscited in this specification are herein incorporated by reference as ifeach individual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method for detecting the presence of at leastone citrus pathogen selected from Citrus tristeza virus (CTV) universal,CTV genotype T30, CTV genotype VT, Citrus psorosis virus, Citrus tatterleaf virus, Citrus leaf blotch virus, Citrus exocortis viroid, Hop stuntviroid, or Citrus leprosis virus in a plant sample, the methodcomprising: extracting RNA from said sample; performing a multiplexbranched DNA signal amplification reaction; wherein the reactioncomprises at least one capture extender probe and at least one labelextender probe that target the pathogen, wherein the at least onecapture extender probe and the at least one label extender probe eachcomprises at least 8, 9, or 10 contiguous nucleotide of a probe sequenceshown in Table 1; and detecting the presence or absence of a signalabove background, wherein the presence of the signal is indicative ofthe presence of the pathogen.
 2. The method of claim 1, wherein thereaction comprises multiple capture extender probes and multiple labelextender probes that target the pathogen, wherein each of the multiplecapture extender probes and multiple label extender probes that targetthe pathogen comprises at least 8, 9, or 10 contiguous nucleotides of aprobe sequence as shown in Table 1; or each of the probes that targetthe pathogen comprises a probe sequence set forth in Table
 1. 3. Themethod of claim 1, further comprising detecting the presence or absenceof a second, third, fourth, fifth, sixth, seventh, or eighth pathogenselected from Citrus tristeza virus (CTV) universal, CTV genotype T30,CTV genotype VT, Citrus psorosis virus, Citrus tatter leaf virus, Citrusleaf blotch virus, Citrus exocortis viroid, Hop stunt viroid, or Citrusleprosis virus in the plant sample, wherein the reaction comprises atleast one capture extender probe and at least one label extender probethat target the second, third, fourth, fifth, sixth, seventh, or eighthpathogen, wherein the capture extender probe and the label extenderprobe that target the second, third, fourth, fifth, sixth, seventh, oreighth pathogen each comprise at least 8, 9, or 10 contiguousnucleotides of a probe sequence as shown in Table
 1. 4. The method ofclaim 3, wherein the reaction comprises multiple capture extender probesand multiple label extender probes that target the second, third,fourth, fifth, sixth, seventh, or eighth pathogen, wherein each of themultiple capture extender probes and multiple label extender probes thattarget the second, third, fourth, fifth, sixth, seventh, or eighthpathogen comprise at least 8, 9, or 10 contiguous nucleotides of a probesequence as shown in Table 1; or each of the probes that target thesecond, third, fourth, fifth, sixth, seventh, or eighth pathogencomprises a probe sequence as shown in Table
 1. 5. The method of claim1, comprising detecting the presence or absence of the nine pathogensCitrus tristeza virus (CTV) universal, CTV genotype T30, CTV genotypeVT, Citrus psorosis virus, Citrus tatter leaf virus, Citrus leaf blotchvirus, Citrus exocortis viroid, Hop stunt viroid, and Citrus leprosisvirus in the plant sample, wherein for each of the nine pathogens, thereaction comprises a capture extender probe and a label extender probe,wherein each capture extender probe and each label extender probecomprises at least 8, 9, or 10 contiguous nucleotides of a probesequence as shown in Table
 1. 6. The method of claim 5, wherein thereaction comprises multiple capture extender probes and multiple labelextender probes that target each of the nine pathogens, wherein each ofthe multiple capture extender probes and each of the multiple labelextender probes comprises at least 8, 9, or 10 contiguous nucleotides ofa probe sequence shown in Table 1; or each of the probes that target thenine pathogens comprises a probe sequence shown in Table
 1. 7. Themethod of claim 1, wherein the reaction comprises at least one blockingprobe listed in Table 1 for the corresponding pathogen; or the reactioncomprises all of the blocking probes listed in Table
 1. 8. The method ofclaim 1, wherein the reaction further comprises using one of the captureextender probes and one of the label extender probes for Nad5 listed inTable 1; or using all of the label extender probes and all of thecapture extender probes for Nad5 listed in Table
 1. 9. The method ofclaim 1, wherein the plant sample is from seed, foliage, limbs, trunk,bark, rootstock, fruit, germplasm, propagule, cuttings, or budwood. 10.A reaction mixture for detecting the presence of at least one citruspathogen selected from Citrus tristeza virus (CTV) universal, CTVgenotype T30, CTV genotype VT, Citrus psorosis virus, Citrus tatter leafvirus, Citrus leaf blotch virus, Citrus exocortis viroid, Hop stuntviroid, or Citrus leprosis virus in a plant sample, wherein the reactionmixture comprises at least one capture extender probe and at least onelabel extender probe that target the pathogen, where the at least onecapture extender probe and at least one label extender probe eachcomprise 8, 9, or 10 contiguous nucleotide of a probe sequence as shownin Table
 1. 11. The reaction mixture of claim 10, wherein the reactioncomprises multiple capture extender probes and multiple label extenderprobes that target the pathogen, wherein each of the multiple captureextender probes and multiple label extender probes comprises at least 8,9, or 10 contiguous nucleotides of a probe sequence as shown in Table 1;or each of the probes that target the pathogen comprises a probesequence set forth in Table
 1. 12. The reaction mixture of claim 10,further comprising at least one capture extender probe and at least onelabel extender probe that target a second, third, fourth, fifth, sixth,seventh, or eighth pathogen selected from Citrus tristeza virus (CTV)universal, CTV genotype T30, CTV genotype VT, Citrus psorosis virus,Citrus tatter leaf virus, Citrus leaf blotch virus, Citrus exocortisviroid, Hop stunt viroid, or Citrus leprosis virus, wherein the captureextender probe and the label extender probe each comprise at least 8, 9,or 10 contiguous nucleotides of a probe sequence as shown in Table 1.13. The reaction mixture of claim 12, wherein the reaction comprisesmultiple capture extender probes and multiple label extender probes thattarget the second, third, fourth, fifth, sixth, seventh, or eighthpathogen, wherein each of the multiple probes comprises at least 8, 9,or 10 contiguous nucleotides of a probe sequence as shown in Table 1; orwherein each of the probes that target the second, third, fourth, fifth,sixth, seventh, or eighth pathogen comprises a probe sequence as shownin Table
 1. 14. The reaction mixture of claim 10, comprising a captureextender probe and a label extender probe for each of the nine pathogensCitrus tristeza virus (CTV) universal, CTV genotype T30, CTV genotypeVT, Citrus psorosis virus, Citrus tatter leaf virus, Citrus leaf blotchvirus, Citrus exocortis viroid, Hop stunt viroid, and Citrus leprosisvirus, wherein each capture extender probe and each label extender probecomprises at least 8, 9, or 10 contiguous nucleotides of a probesequence as shown in Table
 1. 15. The reaction mixture of claim 14,comprising multiple capture extender probes and multiple label extenderprobes that target each of the nine pathogens, wherein each of themultiple capture extender probes and each of the multiple label extenderprobes comprises at least 8, 9, or 10 contiguous nucleotides of a probesequence shown in Table 1; or wherein each of the probes that target thenine pathogens comprises a probe sequence shown in Table
 1. 16. A kitfor detecting the presence of at least one citrus pathogen selected fromCitrus tristeza virus (CTV) universal, CTV genotype T30, CTV genotypeVT, Citrus psorosis virus, Citrus tatter leaf virus, Citrus leaf blotchvirus, Citrus exocortis viroid, Hop stunt viroid, or Citrus leprosisvirus in a plant sample, wherein the kit comprises at least one captureextender probe and at least one label extender probe that target thepathogen, where the at least one capture extender probe and at least onelabel extender probe comprises 8, 9, or 10 contiguous nucleotide of aprobe sequence as shown in Table
 1. 17. The kit of claim 16, wherein thekit comprises multiple capture extender probes and multiple labelextender probes that target the pathogen, wherein each of the multiplecapture extender probes and multiple label extender probes comprises atleast 8, 9, or 10 contiguous nucleotides of a probe sequence as shown inTable 1; or each of the probes that target the pathogen comprises aprobe sequence set forth in Table
 1. 18. The kit of claim 16, furthercomprising at least one capture extender probe and at least one labelextender probe that target a second, third, fourth, fifth, sixth,seventh, or eighth pathogen selected from Citrus tristeza virus (CTV)universal, CTV genotype T30, CTV genotype VT, Citrus psorosis virus,Citrus tatter leaf virus, Citrus leaf blotch virus, Citrus exocortisviroid, Hop stunt viroid, or Citrus leprosis virus, wherein the captureextender probe and the label extender probe each comprise at least 8, 9,or 10 contiguous nucleotides of a probe sequence as shown in Table 1.19. The kit of claim 18, wherein the kit comprises multiple captureextender probes and multiple label extender probes that target thesecond, third, fourth, fifth, sixth, seventh, or eighth pathogen,wherein each of the multiple probes comprises at least 8, 9, or 10contiguous nucleotides of a probe sequence as shown in Table 1 or eachof the probes that target the second, third, fourth, fifth, sixth,seventh, or eighth pathogen comprises a probe sequence as shown inTable
 1. 20. The kit of claim 16, comprising a capture extender probeand a label extender probe for each of the nine pathogens Citrustristeza virus (CTV) universal, CTV genotype T30, CTV genotype VT,Citrus psorosis virus, Citrus tatter leaf virus, Citrus leaf blotchvirus, Citrus exocortis viroid, Hop stunt viroid, and Citrus leprosisvirus, wherein each capture extender probe and each label extender probecomprises at least 8, 9, or 10 contiguous nucleotides of a probesequence as shown in Table
 1. 21. The kit of claim 20, comprisingmultiple capture extender probes and multiple label extender probes thattarget each of the nine pathogens, wherein each of the multiple captureextender probes and each of the multiple label extender probes comprisesat least 8, 9, or 10 contiguous nucleotides of a probe sequence shown inTable 1; or each of the probes that target the nine pathogens comprisesa probe sequence as shown in Table 1.